1. Field of the Invention
The present invention relates to a total reflection illumination apparatus applied to a microscope which illuminates a sample through an objective having a numerical aperture enabling total internal reflection illumination, and a microscope using this total internal reflection illumination apparatus.
2. Description of the Background Art
In recent years, functional analysis of a biological cell has been vigorously carried out. In such function analysis of cells, in order to observe a function of a cell membrane in particular, attention is drawn to a total internal reflection fluorescence microscopy (TIRFM) which acquires total internal reflection fluorescence images from the cell membrane and a nearby part.
In such a total internal reflection fluorescence microscopy (which will be referred to as a “microscope” hereinafter), a total internal reflection illumination which locally illuminates only a sample (which may be also referred to as a “specimen” in some cases) in the vicinity of the glass surface is used. In this microscope, when the illumination light is totally reflected on the interface between the cover glass and the specimen, a fluorescent material is excited by using the light called evanescent light which permeates in a small range of not more than several 100 nm on the specimen side. Therefore, only the fluorescence in a small range in the vicinity of the cover glass is observed. Accordingly, the background (scattered light or the like) is very dark, and the weak fluorescence can be observed (for example, observation of the fluorescence of one molecule of the fluorescent dye).
Meanwhile, in the fluorescence observation by using such a total internal reflection illumination, a permeation depth of the evanescent light which permeates from the glass surface to the sample side varies depending on a refractive index of the glass and others. Further, this permeation depth means a depth along which observation has been carried out, and it also varies depending on a purpose of a speculum user.
Thus, there is also considered varying an incidence angle of the illumination light from the glass to the sample in accordance with conditions of the specimen or a depth along which observation should be carried out.
Meanwhile, in case of performing fluorescence observation using the total internal reflection illumination, there is known a microscope which conducts the total internal reflection illumination which illuminates the sample through an objective.
For example, in such a microscope, a mirror which reflects the light from a light source to an objective side is moved, and an incidence position of the illumination light to the objective is continuously moved in a direction away from the optical axis of the objective. As a result, the incidence angle from the glass to the sample is continuously changed and the incident-light fluorescence illumination and the total internal reflection illumination are switched (see Jpn. Pat. Appln. KOKAI Publication No. 09-159922). It is to be noted that a micrometer or the like is generally used for movement of the mirror which reflects the illumination light, namely, adjustment of the incidence angle from the glass to the sample because fine adjustment is required.
Furthermore, in another microscope, a frame of the objective has a dual structure consisting of an inner frame and an outer frame, the light from the light source is reflected by an annular mirror so that the illumination light can pass between the inner frame and the outer frame in the dual structure. A sample is illuminated with that light, and the return light from the sample is observed through the objective (see Jpn. Pat. Appln. KOKAI Publication No. 10-96861).
In the above-described structure, in the total internal reflection illumination which performs illumination through the objective, the illumination light with which the specimen is irradiated returns to the objective in principle. Moreover, the mirror used to lead the illumination light to the objective side is provided in an observation optical path extending from the objective to observing means.
Therefore, the observation optical path of the fluorescence emitted from the specimen crosses the illumination light or the totally reflected return light. Therefore, the self-fluorescence generated on the illumination light or the totally reflected return light beam enters the observation light beam, and there is a possibility that a fluorescence observation image may be deteriorated.
In addition, since the illumination light and the totally reflected return light also cross each other, an interference fringe may be generated due to crossing of the laser beams when the laser beam is used as the illumination light, for example. An excellent fluorescence observation image can not be obtained due to the influence of the interference fringe.
Additionally, in the microscope disclosed in Jpn. Pat. Appln. KOKAI Publication No. 09-159922, the incident-light fluorescence illumination may be turned on at the time of adjusting the incidence angle from the glass to the sample when performing the fluorescence observation by the total internal fluorescence illumination in some cases. In this case, the mirror must be freshly moved to a position of the total internal reflection illumination. However, since the sample on the glass surface is irradiated with the incident-light illumination with the strong intensity as the exciting light during this movement, the entire sample may lose its color.
Further, although the micrometer or the like is used to move the mirror in the range from the incident-light fluorescence illumination to the total internal reflection illumination, since the micrometer has a small quantity of movement per one rotation of a rotation operation portion, the number of times of rotation increases when switching from the incident-light illumination to the total internal reflection illumination. Therefore, a lot of trouble is taken until this switching, thereby greatly reducing the operability of the fluorescence observation. Furthermore, this means that the entire sample may possibly lose its color during this switching when trying to switch from the incident-light fluorescence illumination to the total internal reflection illumination while irradiating the sample with the illumination exciting light.